The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen conversion catalysts in nature. Their active-site cofactor (H-cluster) comprises a [4Fe-4S] cluster linked to a unique diiron site that binds three carbon monoxide (CO) and two cyanide (CN) ligands. Understanding microbial hydrogen conversion requires elucidation of the interplay of proton and electron transfer events at the H-cluster. We performed real-time spectroscopy on [FeFe]-hydrogenase protein films under controlled variation of atmospheric gas composition, sample pH, and reductant concentration. Attenuated total reflection Fourier-transform infrared spectroscopy was used to monitor shifts of the CO/CN vibrational bands in response to redox and protonation changes. Three different [FeFe]-hydrogenases and several protein and cofactor variants were compared, including element and isotopic exchange studies. A protonated equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially accumulated at acidic pH and under reducing conditions. We show that the one-electron reduced state Hred' represents an intrinsically protonated species. Interestingly, the formation of HoxH and Hred' was independent of the established proton pathway to the diiron site. Quantum chemical calculations of the respective CO/CN infrared band patterns favored a cysteine ligand of the [4Fe-4S] cluster as the protonation site in HoxH and Hred'. We propose that proton-coupled electron transfer facilitates reduction of the [4Fe-4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. Our findings imply that protonation events both at the [4Fe-4S] cluster and at the diiron site of the H-cluster are important in the hydrogen conversion reaction of [FeFe]-hydrogenases.

H2 turnover at the [FeFe]-hydrogenase cofactor (H-cluster) is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydrido-species which again is double-oxidized to release another proton. Three of the four presumed catalytic intermediates (Hox, Hred/Hred and Hsred) were characterized, using various spectroscopic techniques. However, in catalytically active enzyme, the state containing the hydrido-species, which is eponymous for the proposed heterolytic mechanism, has yet only been speculated about. We use different strategies to trap and spectroscopically characterize this transient hydride state (Hhyd) for three wild-type [FeFe]-hydrogenases. Applying a novel set-up for real-time attenuated total-reflection Fourier-transform infrared spectroscopy, we monitor compositional changes in the state-specific infrared signatures of [FeFe]-hydrogenases, varying buffer pH and gas composition. We selectively enrich the equilibrium concentration of Hhyd, applying Le Chatelier’s principle by simultaneously increasing substrate and product concentrations (H2/H+). Site-directed manipulation, targeting either the proton-transfer pathway or the adt ligand, significantly enhances Hhyd accumulation independent of pH.

The unmatched catalytic turnover rates of [FeFe]-hydrogenases require an exceptionally efficient proton-transfer (PT) pathway to shuttle protons as substrates or products between bulk water and catalytic center. For clostridial [FeFe]-hydrogenase CpI such a pathway has been proposed and analyzed, but mainly on a theoretical basis. Here, eleven enzyme variants of two different [FeFe]-hydrogenases (CpI and HydA1) with substitutions in the presumptive PT-pathway are examined kinetically, spectroscopically, and crystallographically to provide solid experimental proof for its role in hydrogen-turnover. Targeting key residues of the PT-pathway by site directed mutagenesis significantly alters the pH-activity profile of these variants and in presence of H2 their cofactor is trapped in an intermediate state indicative of precluded proton-transfer. Furthermore, crystal structures coherently explain the individual levels of residual activity, demonstrating e.g. how trapped H2O molecules rescue the interrupted PT-pathway. These features provide conclusive evidence that the targeted positions are indeed vital for catalytic proton-transfer.

[FeFe]-Hydrogenases contain a H-converting cofactor (H-cluster) in which a canonical [4Fe-4S] cluster is linked to a unique diiron site with three carbon monoxide (CO) and two cyanide (CN) ligands (e.g., in the oxidized state, Hox). There has been much debate whether reduction and hydrogen binding may result in alternative rotamer structures of the diiron site in a single (Hred) or double (Hsred) reduced H-cluster species. We employed infrared spectro-electrochemistry and site-selective isotope editing to monitor the CO/CN stretching vibrations in [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. Density functional theory calculations yielded vibrational modes of the diatomic ligands for conceivable H-cluster structures. Correlation analysis of experimental and computational IR spectra has facilitated an assignment of Hred and Hsred to structures with a bridging hydride at the diiron site. Pronounced ligand rotation during μH binding seems to exclude Hred and Hsred as catalytic intermediates. Only states with a conservative H-cluster geometry featuring a μCO ligand are likely involved in rapid H turnover.

[FeFe]-hydrogenase (H 2 ase) 4 proteins are the most active biological catalysts for the production of molecular hydrogen (H 2 ) from proton reduction, with reported turnover rates of up to 10 4 s Ϫ1 (1, 2). Therefore, these enzymes are of high interest for biotechnology, aiming at the generation of H 2 as a renewable fuel (1, 3-5). However, a severe limitation for such applications is the rapid inactivation of [FeFe]-H 2 ases by dioxygen (O 2 ) (6, 7). Understanding the mechanism of O 2 -induced inactivation may allow the improvement of enzyme features to yield increased O 2 tolerance (e.g. by genetic engineering, which has already been demonstrated for NiFe hydrogenases) (8 -10).[FeFe]-H 2 ases are found in certain bacteria and green algae (11, 12). All of these enzymes contain an active site that consists of an inorganic iron complex, which is denoted as the H-cluster (13-15). The [FeFe]-H 2 ase enzymes from anaerobic bacteria in addition bind several iron-sulfur (FeS) clusters, serving as a relay for electron transfer to and from the active site (13-15).[FeFe]-H 2 ases from green algae represent the minimal unit for biological H 2 production because they contain only the H-cluster, whereas accessory FeS clusters are absent (16). This feature renders these enzymes most suitable for spectroscopic investigations on O 2 -induced inactivation (17), focusing on the active site reactions. The general structure of the H-cluster has been unraveled by crystallography, x-ray absorption spectroscopy (XAS), EPR, and FTIR spectroscopy on H 2 ase protein from various organisms (18). The H-cluster structure in both bacteria and green algae appears to be similar overall (16,19,20). It features a [4Fe4S] cubane cluster, which is bound by four cysteine residues to the protein and is linked by one of them to a binuclear iron unit (2Fe H ) (Fig. 1). The latter carries two cyanide (CN Ϫ ) ligands and three carbon monoxide (CO) ligands (21, 22) and presumably an azadithiolate group (adt; (SCH 2 ) 2 NH) in the metal-bridging position (21,23,24). Both types of diatomic ligands are probably derived from a biosynthetic pathway starting with tyrosine (25-27). The nitrogen atom of the adt has been proposed to be involved in proton transfer at the active site (21, 28). The H-cluster structure is assembled in a complex reaction involving three maturation proteins (29 -34). H 2 formation has been proposed to involve the binding and reduction of a single proton, which transiently creates a hydride ligand, either located in a bridging position between the two iron ions or terminally bound at the distal iron ion of the 2Fe H moiety (Fig. 1). After a second protonation step, H 2 is released from the H-cluster (35)(36)(37) 4 The abbreviations used are: H 2 ase, hydrogenase; adt, azadithiolate; EXAFS, extended x-ray absorption fine structure; NaDT, sodium dithionite; ROS, reactive oxygen species; XANES, x-ray absorption near edge structure; XAS, x-ray absorption spectroscopy; FT, Fourier transform.